Analysis of the function of Sp6 and Sp8 transcription factors in the limb ectoderm

Abstract

We have recently identified the crucial role played by Sp6 and Sp8, two transcription factors members of the Sp family, during limb development. In their absence or substantial reduction no limbs form and their progressive dose reduction associates a spectrum of limb malformations ranking from syndactyly through Split-hand/split-foot malformation, to olygodactyly. When digits form, they exhibit bidorsal tips. We have postulated that Sp6 and Sp8 are necessary, downstream of Wnt/B-catenin, to activate Fgf8 and downstream of Bmp signaling, possible cooperating with Smads, to activate En1, the marker of the ventral ectoderm. Currently we are exploring these suspected protein-protein interactions by co-immunoprecipitation (CoIP) and by bimolecular fluorescence complementation (BiFC). To this end, Sp6, Sp8 and Smads were tagged with Myc or FLAG epitopes to their N-terminal end and with YFP full length or YFP moieties to their C-terminal end. We show by CoIP and BiFC that Sp8 homodimerizes. To determine the region involved in Sp8 interaction, we have generated different truncated versions of Sp8. Our results show that Sp8 homodimerizes at its C-terminal domain. Our study underscores the relevance of Sp6/8 in mediating B-catenin and Bmp signaling pathways in the limb ectoderm.